U.S. patent application number 16/668476 was filed with the patent office on 2020-04-30 for automated method for detecting cervical cancers and high grade hyperplasias.
The applicant listed for this patent is Triantafyllos P. Tsipouras Tafas. Invention is credited to Michael Kilpatrick, Gary Sarkis, Triantafyllos P. Tafas, Petros Tsipouras.
Application Number | 20200131565 16/668476 |
Document ID | / |
Family ID | 39492936 |
Filed Date | 2020-04-30 |
United States Patent
Application |
20200131565 |
Kind Code |
A1 |
Tafas; Triantafyllos P. ; et
al. |
April 30, 2020 |
AUTOMATED METHOD FOR DETECTING CERVICAL CANCERS AND HIGH GRADE
HYPERPLASIAS
Abstract
Automated methods for detecting cancer and related hyperplasias
in biological samples.
Inventors: |
Tafas; Triantafyllos P.;
(Rocky Hill, CT) ; Tsipouras; Petros; (Madison,
CT) ; Kilpatrick; Michael; (West Hartford, CT)
; Sarkis; Gary; (Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tafas; Triantafyllos P.
Tsipouras; Petros
Kilpatrick; Michael
Sarkis; Gary |
Rocky Hill
Madison
West Hartford
Guilford |
CT
CT
CT
CT |
US
US
US
US |
|
|
Family ID: |
39492936 |
Appl. No.: |
16/668476 |
Filed: |
October 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14931833 |
Nov 3, 2015 |
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16668476 |
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11924293 |
Oct 25, 2007 |
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14931833 |
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60862974 |
Oct 25, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 2021/6421 20130101; G01N 21/6458 20130101; G01N 2021/6439
20130101; G02B 21/16 20130101; C12Q 1/6841 20130101; G01N 33/57496
20130101; G02B 21/0004 20130101 |
International
Class: |
C12Q 1/6841 20060101
C12Q001/6841; G01N 21/64 20060101 G01N021/64; G02B 21/00 20060101
G02B021/00; G01N 33/574 20060101 G01N033/574; G02B 21/16 20060101
G02B021/16 |
Claims
1. An automated method of screening for the presence and/or extent
of a pathology in a subject, the pathology characterized by an
abnormal chromosomal component in a cell of the subject, comprising
the steps of a) contacting a biological sample comprising cell
nuclei from said subject with one or more distinguishable labeled
probes directed to at least one chromosomal sequence that
characterizes the abnormality under conditions that promote
hybridization of the one or more probes to the at least one
sequence; b) causing interrogatable information regarding said one
or more distinguishable labeled probes to be associated with said
biological sample; c) automatically obtaining a digitized
representation of the one or more distinguishable labels hybridized
to the chromosomal sequences by irradiating said biological sample
with fluorescence exciting illumination based on said
interrogatable information, capturing one or more images of said
biological sample, digitizing each of said one or more images,
analyzing at least one of said one or more digitizations to cause
adjustment of exposure parameters to form said digitized
representation; d) automatically analyzing the distribution and
intensity of binding of the one or more distinguishable labels in
the digitized representation to determine the presence and/or
extent of an abnormal chromosomal component; and e) automatically
reporting results of the analysis of step d); wherein steps b)-d)
are carried out without intervention by a human.
2. The method described in claim 1 wherein an automated microscope
system carries out steps b)-e).
3. The method described in claim 1 wherein a probe targets at least
one of a single nucleotide polymorphism (SNP), a mutated sequence,
a duplicated or amplified gene or portion thereof, a centromere of
chromosome 3, a centromere of chromosome 7, and a sequence
comprising the TERC gene or a portion thereof.
4. The method described in claim 1 further comprising contacting
the sample under the hybridizing conditions with a tagged reference
probe directed to a chromosomal locus known not to be abnormal, or
contacting the sample with a reference stain, and referencing the
representing and analysis steps to the reference probe or
stain.
5. An automated method of screening for an abnormality related to a
cancer, a high grade hyperplasia or a high grade dysplasia in a
subject, comprising the steps of: a) obtaining a biological sample
comprising nuclei from the subject; b) contacting the nuclei in the
sample with a first probe bearing a first detectable label directed
to a chromosomal sequence related to the abnormality under
conditions that promote hybridization of the probes to targeted
chromosomal loci; c) contacting the sample under the hybridizing
conditions with at least one of a detectably labeled reference
probe directed to a chromosomal locus known not to be abnormal and
a reference stain; d) causing interrogatable information regarding
the first detectable label to be associated with the biological
sample; e) automatically imaging the labels bound to the
chromosomal sequences and imaging the reference stain if used, by
irradiating the biological sample with flourescense exciting
illumination based on the interrogatable information, capturing two
or more images of the biological sample, digitizing each of the two
or more images, analyzing at least two of the two or more
digitizations to cause adjustment of exposure parameters to form
the digitized image of the labels bound to the chromosomal
sequences, and the digitized image of the reference stain; f)
automatically analyzing the two or more digitized images for the
distribution and intensity of hybridized labels and stain if used;
and g) automatically reporting results of the analysis of step f);
wherein steps d)-f) are performed without intervention by a human;
thereby providing an assessment of the abnormality in the
subject.
6. The method described in claim 5 wherein an automated microscope
performs steps d)-g).
7. The method described in claim 5 wherein prior to the contacting
step the nuclei are isolated from the biological sample and are
deposited to form a layer.
8. The method described in claim 5 wherein the sample comprises one
or more of a biopsy, surgical resection specimen, blood, lymph,
urine, an effusion fluid, a biological fluid, an epithelial
scraping, a lavage fluid, aspiration fluid, sputum, and a
tissue.
9. The method described in claim 5 wherein a label is a fluorescent
label.
10. The method described in claim 5 wherein a probe targets at
least one of a single nucleotide polymorphism (SNP), a mutated
sequence, a duplicated or amplified gene or portion thereof a
centromere of chromosome 3, a centromere of chromosome 7, and a
sequence comprising the TERC gene or a portion thereof.
11. An automated method for monitoring the efficacy over time of a
course of therapy in the treatment of a cancer or high grade
hyperplasia in a patient, said method comprising the steps of (a)
obtaining from the patient a fluid biological sample in which cells
associated with the cancer or high grade hyperplasia are found; (b)
treating said fluid biological sample or a portion thereof with one
or more detectably labeled chromosomal probes having a high degree
of sequence similarity to one or more chromosomal loci associated
with, or whose amplification is associated with, said cancer or
high grade hyperplasia, under conditions sufficient to enable
hybridization of said probes to chromosomes in the sample; (c)
automatically scanning said treated fluid biological sample to
obtain a digitized representation of said fluid biological sample
or a portion thereof and detecting said one or more labels bound to
any chromosomes in said sample by irradiating said treated fluid
biological sample with fluorescence exciting illumination based on
the interrogatable information, capturing one or more images of the
biological sample, digitizing each of the one or more images,
analyzing at least one of the one or more digitizations to cause
adjustment of exposure parameters to form the digitized
representation; (d) automatically detecting from the digitized
representation the number of cells associated with said chromosomes
hybridized to said chromosomal probes; and (e) automatically
comparing the hybridization patterns of a label and cell number
results provided in steps (c) and (d) at differing times in the
therapeutic treatment course, thereby evaluating the efficacy of
the therapy in the treatment of the cancer or high grade
hyperplasia.
12. The method of claim 11 wherein monitoring is performed at
intervals of 1 day or greater.
13. The method described in claim 11 wherein the fluid biological
sample comprises one or more of blood, lymph, urine, an effusion
fluid, an epithelial scraping, a lavage fluid, aspiration fluid,
and sputum.
14. The method described in claim 11 wherein an automated
microscope system performs steps (c)-(e) without intervention by a
human.
15. The method described in claim 14 wherein the automated
microscope system automatically optimizes the field scanning the
sample.
16. The method described in claim 14 wherein the automated
microscope scans two or more planes in a field of the sample.
17. The method described in claim 11 wherein a probe targets at
least one of a single nucleotide polymorphism (SNP), a mutated
sequence, a duplicated or amplified gene or portion thereof, a
centromere of chromosome 3, a centromere of chromosome 7, and a
sequence comprising the TERC gene or a portion thereof.
18. The method described in claim 11 wherein a patient obtains
samples without assistance from another human.
19. (canceled)
Description
[0001] This continuation utility patent application claims the
benefit of priority of U.S. Non-Provisional patent application Ser.
No. 11/924,293, filed Oct. 25, 2007, that claims the benefit of
priority of U.S. Provisional Application No. 60/862,974 filed Oct.
25, 2006. This reference and all additional references cited in
this specification, and their references, are incorporated by
reference herein where appropriate for teachings of additional or
alternative details, features, and/or technical background.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention generally relates to an automated
method for detecting cancer, and dysplasias, particularly high
grade dysplasias, in an individual. There is presented in one
aspect a method for monitoring the effectiveness of treatment
protocols in the treatment of one or more cancers.
2. Description of the Related Art
[0003] Many methods are known to aid in the microscopic analysis of
samples. For example, without limitation, it is known that certain
dyes have an affinity for certain cellular or subcellular
structures. Such dyes may therefore be used to aid in analysis by
helping to further elucidate such structures. Binding of dyes to
such structures may be identified and analyzed using various
techniques of microscopic detection.
[0004] Fluorescence microscopy of cells and tissues is well known
in the art. Methods have been developed to image fluorescent cells
in a microscope and extract information about the spatial
distribution and temporal changes occurring in these cells. Some of
these methods and their applications are described in an article by
Taylor, et al. in American Scientist 80 (1992), p. 322-335. These
methods have been designed and optimized for the preparation of a
few specimens for high spatial and temporal resolution imaging
measurements of distribution, amount and biochemical environment of
the fluorescent reporter molecules in the cells. Detection of
fluorescent signals may be by way of an epifluorescent microscope
which uses emitted fluorescent light to form an image (whereas a
conventional reflecting microscope uses scattered illumination
light to form an image). The excitation light of a epifluorescence
microscope is used to excite a fluorescent tag in the sample
causing the fluorescent tag to emit fluorescent light. The
advantage of an epifluorescence microscope is that the sample may
be prepared such that the fluorescent molecules are preferentially
attached to the biological structures of interest thereby allowing
identification of such biological structures of interest.
[0005] Automated methods of conducting microscopic analysis of
biological samples enhance diagnostic procedures and optimize the
throughput of samples in a microscope-based diagnostic facility.
Various co-owned U.S. patent applications, described more fully
below, disclose aspects and embodiments of apparatuses and methods
for automated microscopic analysis. These include an integrated
robotic microscope system, a dynamic automated microscope operation
and slide scanning system, various interchangeable objective
lenses, filters, and similar elements for use in an automated
microscope system, an automated microscope stage for use in an
automated microscope system, an automated microscope slide cassette
and slide handling system for use in an automated microscope
system, an automated microscope slide loading and unloading
mechanism for use in an automated microscope system, automated
methods that employ computer-resident programs to drive the
microscopic detection of fluorescent signals from a biological
sample, useable to drive an automated microscope system, automatic
operation of a microscope using computer-resident programs to drive
the microscope in conducting a FISH assay for image processing.
[0006] The acronym "FISH" (fluorescence in situ hybridization)
references a technique that uses fluorescent tags or labels that
emit a characteristic light or color when illuminated by a light
source, such as an ultraviolet or visible light source, to detect
chromosomal structure. FISH uses fluorescent probes which bind only
to those parts of a chromosome with which they show a high degree
of sequence similarity. Such probes may be directed to specific
chromosomes and specific chromosome regions. The probe has to be
long enough to hybridize specifically to its target (and not to
similar sequences in the genome), but not too large to impede the
hybridization process, and it should be tagged directly with
fluorophores. This can be done in various ways, for example nick
translation and PCR using tagged nucleotides. If signal
amplification is necessary to exceed the detection threshold of the
microscope (which depends on many factors such as probe labelling
efficiency, the kind of probe and the fluorescent dye), probes
labeled with haptens such biotin or digoxygenin are used, and
specific fluorescent tagged antibodies or streptavidin are bound to
the hapten molecules, thus amplifying the fluorescence. The FISH
technique may be used for identifying chromosomal abnormalities and
gene mapping.
[0007] A commonly studied mechanism for gene overexpression in
cancer cells is generally referred to as gene amplification. This
is a process whereby a gene is duplicated within the chromosomes of
an ancestral cell into multiple copies. The process involves
unscheduled replications of the region of the chromosome comprising
the gene, followed by recombination of the replicated segments back
into the chromosome (Alitalo K. et al. (1986), Adv. Cancer Res.
47:235-281). As a result, 50 or more copies of the gene may be
produced. The duplicated region is sometimes referred to as an
"amplicon". The level of expression of the gene (that is, the
amount of messenger RNA produced) escalates in the transformed cell
in the same proportion as the number of copies of the gene that are
made (Alitalo et al.).
[0008] Work with other oncogenes, particularly those described for
neuroblastoma, suggests that gene duplication of the proto-oncogene
is an event involved in the more malignant forms of cancer, and
could act as a predictor of clinical outcome (reviewed by Schwab M.
et al. (1990), Genes Chromosomes Cancer 1:181-193; and Alitalo et
al.). In breast cancer, duplication of the erbB2 gene has been
reported as correlating both with reoccurrence of the disease and
decreased survival times (Slamon D. J. et al. (1987), Science
235:178-182). There is some evidence that erbB2 helps identify
tumors that are responsive to adjuvant chemotherapy with
cyclophosphamide, doxorubicin, and fluorouracil (Muss et al. N Engl
J. Med. 1994 330(18):1260-6).
[0009] Only a proportion of the genes that can undergo gene
duplication in breast cancer have been identified. First,
chromosome abnormalities, such as double minute (DM) chromosomes
and homogeneously stained regions (HSRs), are abundant in cancer
cells. HSRs are chromosomal regions that appear in karyotype
analysis with intermediate density Giemsa staining throughout their
length, rather than with the normal pattern of alternating dark and
light bands. They correspond to multiple gene repeats. HSRs are
particularly abundant in breast cancers, showing up in 60-65% of
tumors surveyed (Dutrillaux B. et al. (1990), Cancer Genet
Cytogenet 49:203-217; Zafrani B. et al. (1992), Hum Pathol
23:542-547). When such regions are checked by in situ hybridization
with probes for any of 16 known human oncogenes, including erbB2
and myc, only a proportion of tumors show any hybridization to HSR
regions. Furthermore, only a proportion of the HSRs within each
karyotype are implicated.
[0010] Second, comparative genomic hybridization (CGH) has revealed
the presence of copy number increases in tumors, even in
chromosomal regions outside of HSRs. CGH is a new method in which
whole chromosome spreads are stained simultaneously with DNA
fragments from normal cells and from cancer cells, using two
different fluorochromes. The images are computer-processed for the
fluorescence ratio, revealing chromosomal regions that have
undergone amplification or deletion in the cancer cells
(Kallioniemi A. et al. (1992), Science 258:818-821). This method
was recently applied to 15 breast cancer cell lines (Kallioniemi A.
et al. (1994), Proc. Natl. Acad. Sci. USA 91:2156-2160). DNA
sequence copy number increases were detected in all 23 chromosome
pairs.
[0011] So, C-K, et al. (Clinical Cancer Research 10: 19-27, 2004)
found internal tandem duplication of cyclic AMP response element
binding protein (CBP), a nuclear transcriptional coactivator
protein, in esophageal squamous cell carcinoma samples from Linzhou
(Linxian), China. So et al. show internal tandem duplication of the
CBP gene is a frequent genetic event in human squamous cell
carcinoma.
[0012] The human epidermal growth factor receptor 2 (HER-2)/neu
(c-erbB-2) gene is localized to chromosome 17q and encodes a
transmembrane tyrosine kinase receptor protein that is a member of
the epidermal growth factor receptor (EGFR) or HER family (Ross, J
S, et al., The Oncologist, Vol. 8, No. 4, 307-325, August 2003).
The HER-2 gene is amplified in a fraction, perhaps 25%, of human
breast cancers.
[0013] Fluorescence in situ hybridization (FISH) is commonly used
for the detection of chromosomal abnormalities including sequence
alterations such as single nucleotide polymorphisms or mutations
found in oncogenes.
[0014] A number of methods and kits have been disclosed for
screening of cancer and dysplasias, such as high grade
dysplasias.
[0015] For example, ProVysion Multi-color Probe Set manufactured by
Abbott Molecular is designed to detect and quantify chromosome 8,
the lipoprotein lipase (LPL) gene located at 8p22, and the C-MYC
gene located at the 8q24 region. Gain of 8q24 and 8p21-22 (LPL) and
loss of heterozygosity are two genetic alterations that have been
observed in abnormal samples. The ProVysion Multi-color Probe Set
consists of three probes with three separate fluorophore labels.
The multicolor probe set design is said to permit simultaneous
analysis of the three genomic markers within a single cell,
CEP.RTM. 8 probe labeled with SpectrumAqua, LSI LPL labeled with
SpectrumOrange, and LSI C-MYC labeled with SpectrumGreen. The CEP 8
alpha satellite DNA probe hybridizes to the centromere region of
chromosome 8 (8 p11.1-q11.1) and provides a mechanism for the
identification of copy number of chromosome 8. The LSI LPL
hybridizes to the LPL gene at 8p22 and is approximately 170 kb in
size. The LSI C-MYC Probe (an approximately 750 kb probe)
hybridizes to the C-MYC gene located at 8q24. The manufacturer
asserts that in a normal cell hybridized with the ProVysion
Multi-color Probe Set, the expected pattern is the two orange, two
green and two aqua (202G2A) signal pattern, while in an abnormal
cell, combinations of copies of the three probe signals may be
observed. The test kit indicates that copy numbers of more or less
than two of any probe indicates chromosome or gene gain or loss,
respectively. Less than two copies of the LSI LPL or multiple
copies of the LSI C-MYC Probe relative to CEP 8 copy number
indicates loss of the LPL region and gain of the C-MYC region,
respectively, relative to the chromosome 8 copy number.
[0016] U.S. Patent Publication Nos. 2004/028107 and 2005/0026190 to
Vysis, Inc. assert methods of using probes and probe sets for the
detection of high grade dysplasia and carcinoma in cervical cells.
The methods entail hybridizing one or more chromosomal probes to a
biological sample and detecting the hybridization pattern of the
chromosomal probes to determine whether the subject has high grade
dysplasia or carcinoma. The methods encompass the use of a set of
one or more probes demonstrating a vector value of about 60 or less
wherein the vector value is calculated by
Vector=[(100-specificity).sup.2+(100-sensitivity).sup.2].sup.1/2
The chromosomal probes may comprise probes for specific loci, such
as 8q24, 3q36, Xp22, and CEP 15, or probes, for example,
substantially complementary to full coding sequence for each of
HPV-16, HPV-18, HPV-30, HPV-45, HPV-51, and HPV-58. The biological
sample screened may be pre-screened for the presence of a cell
cycle protein, such as p16 or Cyclin E, or a cell proliferation
marker, such as protein Ki67 or PCNA.
[0017] U.S. Patent Publication 2006/0063194 to Abbott Molecular
also discloses probe sets and methods of using probes and probe
sets for the detection of cancer, particularly lung cancer. Locus
specific probes and chromosome enumeration probes are used in
conjunction, and the hybridization pattern of the same used to
determine whether the subject has lung cancer. Chromsomal
compositions are specified, for example, a probe set for
determining lung cancer may comprise a 5p15 locus specific probe, a
8q24 locus specific probe, a chromosome 6 enumeration probe and a
7p12 locus specific probe.
[0018] Diagnostic FISH light dot counting has been conventionally
performed manually, by a skilled microscopist. In addition to
correctly identifying the dot and its color, other size and shape
characteristics must be categorized to correctly identify the
chromosomal condition. The analysis is made more difficult by the
time constraints imposed by the phenomena. The microscopist,
therefore, must be trained to perform the examination. Even under
the best conditions, the process has proven to be tedious,
lengthily and subject to human error.
[0019] The application of automated microscopy has the potential to
overcome many of the shortcomings of the manual approach. The
automatic microscope can reliably identify the fluorescent dots in
a sample, accurately determine their color, categorize them based
on shape and size, and perform the summary analysis necessary to
determine the presence or absence of the targeted condition without
the inevitable subjective factors introduced by a human operator
all in a timely manner.
[0020] It should be noted that kits for the detection of cancers
are typically designed to provide only a positive or negative
answer--one has a particular cancer or not. While such tests may
indicate the need for interventional therapy, such as chemotherapy,
they are not designed to lead one in the direction of the most
appropriate interventional therapy. Rather, cancer patients are
often subjected to multiple therapies, and the effectiveness of
therapies determined by snap-shots of the cancer status at points
in time after start of the therapy. Such snap-shots may entail for
example, MRI and CAT scans of the body to determine the growth or
shrinkage of tumors. As such snap-shot methods may entail
considerable economic costs, as well as risks in themselves (e.g.,
radiation exposure), such snap-shots may be taken at considerably
longer intervals than might be desired given the need for rapid
intervention into resolving the disease state. As set forth below,
the present inventors have also recognized that the use of
automated microscopy may also be used advantageously to determine
not only whether a person is inflicted with a particular
cancer/hyperplasia, but also as a monitoring tool for the
determination of the efficacy of different interventional
therapeutic approaches to the treatment of cancer/high grade
hyperplasia. In one embodiment, the monitoring of therapeutic
efficacy is by means of monitoring cancer/hyperplastic cells in the
systemic circulation (including the vasculature and lymph system)
with a decrease in number of abnormal cells associated with the
cancer/hyperplasia being taken as an indication of therapeutic
success, and the degree of reduction in such cells being used as a
gauge of the efficacy of one therapy against another therapy.
[0021] There remains a need in the field for the automated imaging
and analysis of images arising from cancer tissue samples treated
with detectably labeled probes, including fluorescently labeled
probes. Additionally there remains a need for convenient, rapid,
hands-free automated fluorescence microscopy of such labeled
samples.
SUMMARY OF THE INVENTION
[0022] Various embodiments are disclosed herein.
[0023] In one embodiment, an automated method of screening for the
presence and/or extent of a pathology in a subject, the pathology
characterized by an abnormal chromosomal component in a cell of the
subject, comprising the steps of
[0024] a) contacting a biological sample comprising cell nuclei
from said subject with one or more distinguishable labeled probes
directed to at least one chromosomal sequence that characterizes
the abnormality under conditions that promote hybridization of the
one or more probes to the at least one sequence;
[0025] b) automatically obtaining a representation of the one or
more distinguishable labels hybridized to the chromosomal
sequences;
[0026] c) automatically analyzing the distribution and intensity of
binding of the one or more labels in the representation to
determine the presence and/or extent of an abnormal chromosomal
component; and
[0027] d) automatically reporting results of the analysis of step
c);
[0028] wherein steps b)-d) are carried out without intervention by
a human
[0029] In various further embodiments of the method of screening an
automated microscope system carries out one, and usually all, of
the steps of automatically obtaining a representation,
automatically analyzing binding, and automatically reporting
results. In this method obtaining the representation and performing
automated image analysis identifies nucleic acid properties
characteristic of a pathology. Various targeted chromosomal
abnormalities may include a single nucleotide polymorphism (SNP), a
mutated sequence, or a duplicated gene or portion thereof.
Chromosomal targets for a probe may include a centromere, or a
target sequence of human chromosome 3 or human chromosome 7, and
all or part of a TERC gene. In additional embodiments various
reference probes directed to a chromosomal locus known not to be
abnormal or a reference stain may be used such that the
representing and analysis steps are referenced to the reference
probe or stain.
[0030] In an additional embodiment an automated method of screening
for an abnormality related to a cancer, a high grade hyperplasia or
a high grade dysplasia in a subject, comprising the steps of:
[0031] a) obtaining a biological sample comprising nuclei from the
subject,
[0032] b) contacting the nuclei in the sample with a first probe
bearing a first detectable label directed to a chromosomal sequence
related to the abnormality under conditions that promote
hybridization of the probes to targeted chromosomal loci;
[0033] c) contacting the sample under the hybridizing conditions
with at least one of a detectably labeled reference probe directed
to a chromosomal locus known not to be abnormal and a reference
stain;
[0034] d) automatically imaging the labels bound to the chromosomal
sequences, and imaging the stain if used;
[0035] e) automatically analyzing an image for the distribution and
intensity of hybridized labels and stain if used; and
[0036] f) automatically reporting results of the analysis of step
e);
[0037] wherein steps d)-f) are performed without intervention by a
human; thereby providing an assessment of the abnormality in the
subject.
[0038] In an embodiment of this method of screening the nuclei are
isolated from the sample, and the nuclei are deposited to form a
layer of nuclei prior to the contacting step. In further
embodiments an automated microscope performs at least one, and
usually all, steps of automatic imaging, automatic analyzing, and
automatic reporting of results. The single layer nuclei preparation
may be obtained by a number of methods known in the art, for
example, by appropriate processing of thin sections from
paraffin-embedded tumor tissue samples. A large variety of origins
for the sample obtained from the subject is envisioned in this
method. In additional embodiments of this method of screening an
automated microscope is used at various stages of the method,
including to automatically provide the images, to obtain the image
the microscope automatically optimizes the field in which the image
occurs, and to obtain images from two or more planes in a field of
the sample to perform the automatic analysis of the image. In
various embodiments the abnormality may be a cancer, a high grade
hyperplasia or a high grade dysplasia. Various abnormalities
targeted by the probe may include a single nucleotide polymorphism,
a mutated sequence, or a duplicated gene or portion thereof.
Additionally in certain embodiments the probe targets a centromere
of chromosome 3 or a centromere of chromosome 7, or a sequence that
includes the TERC gene or a portion thereof.
[0039] In still a further embodiment an automated method for
monitoring the efficacy over time of a course of therapy in the
treatment of a cancer or high grade hyperplasia in a patient is
disclosed. This method includes the steps of
[0040] (a) obtaining from the patient a fluid biological sample in
which cells associated with the cancer or high grade hyperplasia
are found;
[0041] (b) treating the fluid biological sample or a portion
thereof with one or more detectably labeled chromosomal probes
having a high degree of sequence similarity to one or more
chromosomal loci associated with, or whose amplification is
associated with, the cancer or high grade hyperplasia, wherein the
treating is carried out under conditions sufficient to enable
hybridization of the probes to chromosomes in the sample;
[0042] (c) automatically scanning the treated fluid biological
sample and detecting the one or more labels bound to one or more
chromosomal probes that are hybridized to any chromosomes in the
sample;
[0043] (d) automatically detecting the number of cells associated
with the chromosomes hybridized to said chromosomal probes; and
[0044] (e) automatically comparing the hybridization patterns of a
label and cell number results provided in steps (c) and (d) at
differing times in the therapeutic treatment course, thereby
evaluating the efficacy of the therapy in the treatment of the
cancer or high grade hyperplasia.
[0045] In an embodiment the monitoring is performed at intervals of
1 day, or longer. In various embodiments of this method for
monitoring efficacy the fluid biological sample includes one or
more of blood, lymph, urine, an effusion fluid, an epithelial
scraping, a lavage fluid, aspiration fluid, and sputum. In further
embodiments of this method for monitoring efficacy an automated
microscope system performs at least one of the automatic scanning
and the automatic detection uses an automated microscope system, as
well as automatically optimizes the field scanning the sample, and
further scans two or more planes in a field of the sample. In
various embodiments the automated microscope system operates
without intervention by a human. In still additional embodiments a
probe targets at least one of a single nucleotide polymorphism
(SNP), a mutated sequence, a duplicated or amplified gene or
portion thereof, a centromere of chromosome 3, a centromere of
chromosome 7, and a sequence comprising the TERC gene or a portion
thereof.
[0046] In still further embodiments a method for the automated high
throughput characterization of a chromosomal abnormality is
disclosed. This method includes the steps of:
[0047] a) providing at least one microscope slide comprising a
biological sample thereon, wherein the sample is suspected of
harboring the chromosomal abnormality and wherein the sample has
been hybridized to at least one detectably labeled probe specific
for detection of the abnormality,
[0048] b) installing the at least one sample-bearing slide in a
means for automated, reversible, placement of the slide on the
stage of an automated microscope;
[0049] c) causing the placement means automatically and reversibly
to place a sample-bearing slide to be reversibly placed on the
microscope stage;
[0050] d) causing the microscope automatically to obtain at least
one image of the specimen wherein the image comprises a
representation of a labeled probe hybridized to a chromosome;
[0051] e) causing the microscope automatically to analyze the image
in order to characterize the abnormality;
[0052] f) automatically reporting the results of the analysis of
step (e); and
[0053] g) automatically repeating steps (c)-(f).
[0054] In various embodiments the automated microscope operates
without intervention by a human. In additional embodiments of this
high throughput method the automatic microscope obtains an image
automatically by optimizing the field in which the image occurs,
and obtains images from two or more planes in a field of nuclei.
The biological sample may originate in any of various tissues and
biological fluids. Furthermore the abnormality may be a cancer, a
high grade hyperplasia or a high grade dysplasia. Various
abnormalities targeted by the probe used in the high throughput
method may include at least one of a single nucleotide polymorphism
(SNP), a mutated sequence, a duplicated or amplified gene or
portion thereof, a centromere of chromosome 3, a centromere of
chromosome 7, and a sequence comprising the TERC gene or a portion
thereof.
[0055] A further embodiment discloses a method that includes in
order: (a) hybridizing to a biological sample one or more
chromosomal probes having a high degree of sequence similarity to
one or more portions of chromsomic material under conditions
sufficient to enable hybridization of the probes to chromosomes in
the sample (if any), the probes characterized in being tagged with
one or more tags detectable by a detector; (b) automatically
scanning the biological sample and detecting by a detector the one
or more tag(s) associated with the one or more chromosomal probes
that is hybridized to any chromosomes in the sample; and (c)
automatically reporting chromosomes if any in the sample which are
tagged with hybridized probe and the particular probes associated
with the chromosome.
[0056] In various embodiments of the methods disclosed herein the
centromeric probe may be directed to chromosomes known to house
loci the replication of which, or the existence of which, are
associated with a particular cancer state. For example, the
centromeric probe may be direct to chromosome 3 and/or chromosome
7. The locus specific probe may be for single copy sequences may
likewise hybridize with loci associated with cancer, such as loci
on the q am of chromosome 3. The probe itself may advantageously
have a high degree of sequence similarity to one or more portions
of chromosomal material associated with a locus associated with, or
the amplification of which is associated with, particular
cancer(s)/hyperplasias under conditions sufficient to enable
hybridization of the probes to chromosomes in the sample. The
probe, for example, may be a contig consisting of four overlapping
BAC clones containing the TERC gene at chromosomal location 3q26.
Additional centromeric or locus specific probes may be added to a
probe mixture. The nuclear staining may be by way of counterstain
process. The nuclear stain may be, for example, DAPI. In
automatically scanning the sample, the sample may be loaded onto an
automated microscope which automatically moves from one field of
view to another. The microscope may be programmed or otherwise
operationally configured to allow monitoring of a number of signal
channels. For example, an automated microscope may scan in DAPI and
other fluorescence channels (to enumerate, for example, signals for
chromosome 3, locus on 3q, and other centromeric or locus specific
signal). The scanned nuclei may be automatically recorded by the
automated microscope, and/or may be presented to a cytogeneticist
and/or pathologist, or other health care provider. Presentation may
be in numerous fashions, such as in a sorted manner with the ones
with the abnormal counts presented first (e.g., counts not equal to
2 of the 3q being present first). Different cancers may be
detected, such as cervical cancer (using, for example, the
centromeric probe for chromosome 3 and/or chromosome 7 and a locus
specific probe for single copy sequences on the q arm of chromosome
3 comprising a contig consisting of four overlapping BAC clones
containing the TERC gene at chromosomal location 3q26 or a portion
thereof, a DAPI nuclear counterstain, and then enumerating signals
for chromosome 3, locus on 3q, and finding abnormal counts of not
equal of 2 of the 3q related signals).
[0057] Automatic scanning in such embodiments may be performed, for
example, by an automated microscope wherein the biological specimen
is placed on slides which are manually or automatically loaded onto
the microscope stage, and the slide automatically scanned.
Automated microscopes that may find employment in such system are
such as described in other of applicant's patent applications (see
below). Scanning may also be made of other substrates onto/into
which the biological sample is placed. Scanning may comprise
scanning the biological sample in one plane, or in more than one
plane, such as, for example, two, three or more planes. By scanning
in multiple planes, detection of abnormal cells, which may be rare
in terms of total number of cells in the sample, may be
significantly improved. The probes may make use of FISH probes in
which the fluorescent signal is picked up by the detector. The
probes may produce a signal with or without another input signal,
e.g. they may be radioactive, or fluoresce when impinged by an
activating signal (such as an appropriate wavelength of light or
other electromagnetic radiation). The probes may be directed to
different replication associated cancer loci, and may comprise
different fluorescent tags so as to produce different signals. The
detector may be selected in accordance with signal(s) which are to
be produced by the tags, e.g. a fluorescence detector for detecting
fluorescent tags, with the detector operatively configured to
permit detection of the particular fluorescent signals produced by
the fluorescent tags. Reporting may include a simple report of the
particular tags associated with the particular chromosome and/or
may comprise an automatic diagnostic (indicating the type of cancer
associated with the particular hybridization pattern of the
chromosomes). The vector value as compared to normal specimens may
be selected to be less than a particular threshold, such as less
than about 60, less than about 40, less than about 30, less than
about 20, less than about 10, or less than about 0.500. A useful
system may comprise automatic scanning and detection in multiple
signal channels at once, or in a relatively short period of time
(e.g., less than 1 minute) from one another. The system may be
operatively configured to process each of the multiple signals in
real time, simultaneously or concurrently (or a mix of the same),
to allow for quick detection of chromosomal regions, and/or
regional replications, which are indicative of one or more
particular cancer/hyperplasia.
DETAILED DESCRIPTION OF THE INVENTION
[0058] As used herein, "tag" and "label" relate synonymously to a
moiety conjugated to a probe to render the probe detectable by a
particular detection method and modality.
[0059] As used herein "probe" relates generally to a substance
specifically designed to bind to a cellular target, and not to bind
significantly to cellular moieties or structures not intended to be
a target. In several embodiments a probe may be a nucleic acid,
polynucleotide or oligonucleotide whose sequence is sufficiently
complementary to a target sequence in a cellular chromosome or
other nucleic acid to hybridize to the latter structure under
appropriate conditions. In various additional embodiments a probe
may be an antibody or a portion thereof bearing a specificity
determining binding site that specifically targets a cellular
structure.
[0060] As used herein "representation" relates generally to any
visual, graphical, numerical, or similar assembly of information
that characterizes a result obtained using a particular detection
method to examine a biological sample. By way of nonlimiting
example, a representation includes an image of a microscopic field
that includes at least a portion of a biological sample, an image
further modified for example by computer driven means to convey
information by attaching color values to particular features in a
field, a graphical presentation characterizing particular features
derived from an image of a sample, and a table of values or verbal
entries characterizing features derived from an image.
[0061] As used herein "target", "targeted", "targeting" and similar
words or phrases relate generally to a cellular structure to which
a probe is specifically directed. A target is any structure or
component that is a member of a specific binding pair constituted
of the probe and the target. The probe and target have high
specificity and affinity for binding to each other, and low
specificity and low affinity for a probe, or for a target,
respectively, not intended to be recognized. For a probe that
includes a nucleic acid or at least a specific sequence of bases, a
target is a complementary sequence found in chromosomal or nucleic
acid components of a cell. For a probe that is an antibody or
specific binding fragment thereof, a target may be an antigenic or
hapten structure found in a cell. In this framework, a probe is a
"targeting" moiety, and the target structure is "targeted" by the
probe.
[0062] There are provided herein systems and methods for detecting
and monitoring cancers and hyperplasias, particularly high grade
hyperplasias, employing automated detection of signals.
[0063] In a representative embodiment, a biological sample is
interrogated with one or more chromosomal probes having a
detectable tag. The chromosomal probes may be selected and/or
configured to have a high degree of sequence similarity to one or
more portions of chromosomal material which is indicative of an
element associated with a cancer or hyperplasia such as a high
grade hyperplasia. The probes may be selected such that they
associate with regions on the chromosome which are indicative of a
cancer/hyperplasia or the amplification of which is associated with
a cancer/hyperplasia. For example, multiple replications of a
particular loci on a chromosome may be indicative of a
cancer/hyperplasia. The tag on the probes advantageously is
detectable either directly or indirectly (e.g. by binding of
another detectable molecule to a portion of the tag). In one case,
the tag is fluorescent, such as in FISH (fluorescent in situ
hybridization). To promote hybridization between the tagged probe
and the loci of interest on the chromosomal material, hybridization
should be conducted under conditions sufficient for hybridization.
In such embodiment, the sample is automatically scanned using a
detector that can detect the tags. Automatic scanning may be by
means of an automated microscope which is operatively configured to
search a sample through multiple fields of view without the need
for human intervention. The ability to associate the tags with
particular chromosomes enables one to determine whether the
hybridization profile is indicative of a cancer or hyperplasia,
such as a high grade hyperplasia. Such association permits a
determination of whether a cancer/hyperplasia is likely there.
Optionally, the system of such embodiment may include a means, for
example software, hardware, or a software/hardware combination, for
automatically reporting chromosomes in the sample which are tagged
with the hybridized probe and the particular probes that are
associated with the chromosome. Automatic diagnosis based upon the
hybridization may also be provided as part of the automated
microscope system.
[0064] In another representative embodiment, there is provided a
method and system for monitoring the efficacy of a therapy to treat
a cancer or hyperplasia, such as a high grade hyperplasia. The
monitoring can be conducted over time in order to trace the effect
of the therapeutic regimen as the patient is being treated. In such
embodiment, a readily available fluid sample, such as blood, lymph,
and effusion fluid, a lavage fluid, or an aspiration fluid, is
taken from a patient under therapy for treating the cancer and/or
hyperplasia. Any sample so obtained has in it nucleated cells,
including cells suspected of harboring a detectable chromosomal
abnormality characteristic of a cancer or high grade hyperplasia.
The sample is then treated with chromosomal probes that hybridize
with specific loci or positions in the chromosomal material, for
example to detect amplification associated with an abnormal sample,
comprised by the fluid sample. Optionally multiple probes directed
to different loci two or more of which are associated with a
particular cancer/hyperplasia may be used. Use of such combinations
may improve the efficiency of the detection of the
cancer/hyperplasia. Such multiple probes are advantageously tagged
with different tags, such as different fluorescent tags. The tags
are selected to be readable by the detector associated with an
automated scanning device, such as an automated microscope, which
is operatively configured to repeatedly view discrete areas of the
sample without human intervention. By detecting the tags associated
with one or more chromosomal probes that are hybridized to
chromosomes in the sample, one can determine if a hybridization
pattern indicative of a cancer/hyperplasia is seen. Improvement may
be had by automatically detecting the number of cells associated
with the chromosomes hybridized by the chromosomal probes. That is,
by judging whether the number of cells in the fluid indicative of
an abnormal chromosomal complement is lower or higher than the
number of cells seen at an earlier time, one may decide whether the
therapy being used is being effective in the treatment of the
cancer/hyperplasia being treated. The efficacy of a particular
defined therapy on a particular cancer is thus based on changes in
the number of cells detected in the biological sample over time.
For example, if less cells are seen after treatment than before, it
may be determined that the therapy is working. Different therapy
may also be compared by the degree of reduction seen in such
circulating abnormal cells.
[0065] In a variant of a method provided a method and system for
monitoring the efficacy of a therapy to treat a cancer or
hyperplasia, a patient may provide samples independently of visits
to a medical or hospital facility. For example, a patient may be
provided with a kit, a system, or similar equipment for obtaining a
sample of blood for subsequent analysis by methods described
herein. In such nonlimiting examples, a small volume of sample
blood, such as one drop or a few drops, are harvested, optionally
treated to prevent clotting, optionally disposed on a slide, or
otherwise maintained in a state suitable for subsequent analysis.
The scheduling of accumulating such samples may include daily
sampling, or sampling every other day, or twice weekly, or weekly,
or biweekly, or monthly, or at even greater intervals. Samples may
be stored in desiccated chambers, and may be refrigerated or frozen
while awaiting shipment or transfer, and subsequent analysis.
[0066] There is also described in a representative embodiment, a
system/method that can be used for detection of
cancers/hyperplasias, such as high degree hyperplasias that are
related with the amplification of chromosome 3q. In an embodiment
method, a single layer preparation of nuclei for interphase FISH
hybridization is made. For example, the layer of nuclei may be
obtained following appropriate processing of thin sections from
paraffin-embedded tumor tissue samples to provide a nuclear smear.
The nuclear smear is then stained using a centromeric probe for
chromosome 3 or chromosome 7. Subsequently, previously, or
concurrently, the nuclei smear is also stained with a locus
specific probe for single copy sequences on the q arm of chromosome
3 which are indicative of a cancer/hyperplasia state of interest.
For example, the probe can be a contig consisting of four
overlapping BAC clones containing the TERC gene at chromosomal
location 3q26 or a portion thereof. Other centromeric or locus
specific probes can be added to the probe mixture. In one
advantageous aspect, each probe is labeled with a different
fluorochrome to allow for easier detection of distinct signals.
Optionally the smear may be counterstained with a nuclear stain,
such as DAPI. The stained smear is then applied to an automated
scanning device, such as an automated microscope, and automatically
scanned in DAPI and as many fluorescence channels as needed to
enumerate signals for chromosome 3, locus on 3q and any other
centromeric or locus specific signal. The scanned nuclei may be
presented to a health care profession, such as a cytogeneticist or
pathologist for review thereof. The presentation to the health care
profession may be in a sorted manner, for example with the nuclei
with abnormal counts (e.g., not equal to 2) of the 3q related
signals being presented first. Alternatively, or in conjunction,
the system may be operatively configured (e.g. by means of a
program) to analyze the scanned nuclei based on pre-programmed
algorithms (for example) and to provide an automated diagnostic
indication to the health care provider. Such test may be use for
the detection of a number of cancers, including cervical
cancer.
[0067] Automated apparatuses and methods for carrying out the
microscopic analysis of biological samples enhance diagnostic
procedures and optimize the throughput of samples in a
microscope-based diagnostic facility. A robotic microscope system
is described in co-owned U.S. patent application Ser. No.
11/833,203 filed Aug. 2, 2007. Among its disclosures, an integrated
microscope system displaceable along a second surface is provided.
The integrated microscope system includes an automated robotic
microscope system housed in a light-tight enclosure. In this
system, the automated robotic microscope system includes (i) a
microscope having a stage; (ii) at least one specimen slide
positionable on the stage; (iii) a light source that illuminates
the slide; (iv) an image capture device that captures an image of
the specimen; and (v) electrical, electronic and/or computer-driven
means communicating with and controlling positioning of said
specimen slide, said light source, and said image capture device.
Furthermore, in this system the light-tight enclosure includes at
least one shelf interior to said enclosure, wherein said automated
robotic microscope system is positioned on a shelf; and a viewing
monitor capable of displaying images or representations of a
microscopic field being viewed or analyzed that is disposed in a
surface of said enclosure viewable from a location exterior to the
enclosure.
[0068] A dynamic automated microscope operation and slide scanning
system is described in co-owned U.S. patent application Ser. No.
11/833,594 filed Aug. 3, 2007. Embodiments disclosed include an
automated microscope and method for dynamically scanning a specimen
mounted on a microscope slide using a dynamic scanning microscope
incorporating a microscope slide stage, at least one source of
illumination energy, at least one electronic imaging device, at
least one interchangeable component carousel and a synchronization
controller. An exemplary automated microscope has the ability to
significantly reduce the time required to perform an examination,
reduce vibration reaching the system, and to provide diagnostic
results. During the imaging process, the stage and color filter
wheel are in constant motion rather than stationary as in previous
approaches. Real time position sensors on each of the moving
sub-systems accurately telemeter the instant position of the stage
mounted slide and the color filter wheel. The color filter wheel
rotates at a sufficient speed to allow the capture of images, at
each of the filter wavelengths, at each imaging location and focal
plane.
[0069] Interchangeable objective lenses, filters, and similar
elements for use in an automated microscope system are described in
co-owned U.S. patent application Ser. No. 11/833,154 filed Aug. 2,
2007. This application generally relates to remotely operated or
robotically controlled microscopes, and specifically to the
mechanization of a means for automatically interchanging objective
lens assemblies, filters and/or other optical components. An
apparatus for interchanging optical components in an optical path
is disclosed, which includes a control motor having a rotatable
motor shaft; a support structure supporting the control motor; a
planar base defined by a periphery that is generally symmetric
about a central point on the planar base, the planar base including
a plurality of mounting fixtures housing a plurality of optical
components equi-angularly placed at a same distance from the base
center, and a mechanism that causes generally symmetric rotation of
the planar base about its center, so that a particular optical
component of choice is positioned in the optical beam.
[0070] An automated microscope stage for use in an automated
microscope system is described in co-owned U.S. patent application
Ser. No. 11/833,183 filed Aug. 2, 2007. This application generally
relates to a microscope stage that is adjustably moveable along the
optic axis of the microscope. For example, a microscope slide mount
is disclosed that is adjustable along a direction of the optic axis
of the microscope, including a base plate; a microscope stage
assembly movably mounted on said base plate operably configured to
permit displacement of the assembly along the direction of the
optic axis; and a microscope slide holding means fixed to said
microscope stage assembly.
[0071] An automated microscope slide cassette and slide handling
system for use in an automated microscope system is disclosed in
co-owned U.S. patent application Ser. No. 11/833,517 filed Aug. 3,
2007. This application discloses a mechanism for removing and
replacing a slide housed in a cassette defining a plurality of
slots configured for holding slides in spaced parallel
configuration.
[0072] An automated microscope slide loading and unloading
mechanism for use in an automated microscope system is described in
co-owned U.S. patent application Ser. No. 11/833,428 filed Aug. 3,
2007. An exemplary embodiment discloses a microscope slide
manipulation device which includes: a base structure; a sleeve
defining a through-void, the sleeve having a first end and a second
end, the second end fastened to the base, and the sleeve being
oriented perpendicular to the base; a longitudinal shaft symmetric
about an imaginary longitudinal axis in part positioned in the
sleeve through-void in a manner to permit axial and longitudinal
movement of the longitudinal shaft in the sleeve through-void, the
longitudinal shaft having a shaft first end and a shaft second end,
the shaft second end positioned within the sleeve through-void and
the shaft first end projecting beyond the sleeve first end and
including a parallel track structure in a plane to the sleeve
imaginary longitudinal axis; a plate slideably positioned between
the parallel track structures on the sleeve first end, the plate
having a first plate end and a second plate end, one of the first
plate end or second plate end having a two-pronged forked
configuration defining a void area between each prong that
corresponds to the width of a microscope slide, and wherein the
fork has a gripping structure operatively configured to permit
gripping of a microscope slide along its edges.
[0073] Automated methods that employ computer-resident programs to
drive the microscopic detection of fluorescent signals from a
biological sample, useable to drive an automated microscope system,
are disclosed in co-owned U.S. patent application Ser. No.
11/833,849 filed Aug. 3, 2007. An exemplary method of microscopic
analysis, adaptable for high throughput analysis of multiple
samples, disclosed therein includes steps of providing an automated
microscope comprising a slide stage, at least one objective lens,
image capturing means, programmable means for operating the
microscope according to a protocol, and programmable means for
providing an analytical outcome; providing a microscope slide
containing a sample and interrogatable data thereon, wherein the
interrogatable data provide information related to a protocol for
analysis of said sample; interrogating the data; positioning the
slide on the slide stage; causing the microscope to analyze the
sample in accordance with the analytical protocol encoded in the
interrogatable data; and causing the microscope to provide an
analytical outcome representing the sample. Automatic operation of
a microscope using computer-resident programs to drive the
microscope in conducting a FISH assay for image processing is
described in co-owned U.S. patent application Ser. No. 11/833,204
filed Aug. 2, 2007. Embodiments are disclosed which perform various
image processing functions that may be employed to implement an
automated fluorescence in situ hybridization method. The
embodiments include an auto-exposure method for acceptably imaging
all regions of the sample over an intensity range exceeding the
dynamic range of the digital electronics; a method for enumeration
of fluorescence in situ hybridization objects-of-interest which
locates targets within the sample; nuclei identification which is a
method for classifying and characterizing the objects-of-interest
enumerated; segmenting nuclei which, is a method for defining the
shape of an identified object of interest. Embodiments of the
method are useful to characterize cell nuclei, or to enumerate a
chromosome.
[0074] The automated microscope system described in the preceding
paragraphs operates under control of computer-resident and
computer-implemented instructions. Accordingly the system permits
automated detection and analysis of samples without human
intervention. The automated slide cassette and automated slide
loading and unloading mechanism permit unattended high throughput
analysis of a plurality of samples.
[0075] Methods disclosed herein are directed toward automating the
detection and analysis of tissue specimens whose cells are
suspected of harboring genes that have undergone somatic gene
duplication or gene amplification during carcinogenesis. The
methods afford computer driven image accumulation, and computer
driven analysis of images obtained, as well as reporting results of
such analyses in a variety of formats in an automated procedure
that frees the methods from human intervention to a significant
extent. Reports may be presented, by way of nonlimiting example, in
the form of charts, tables, images of representations of a field on
a slide, and the like. Reports are in digital formats as files or
records, and as such are conveniently disseminated to local or
remote locations for review. Because of the use of automated
fluorescence microscopy, such as a system including components and
software that is referenced herein, rapid, convenient, and accurate
screening of tissue samples is afforded. These methods, and the
automated microscope system employed in implementing them, are
particularly well suited for use in high throughput analysis of a
plurality of tissue samples.
[0076] Tissue samples may be derived from medical or surgical
procedures that yield specimens from suspect tissues or organs,
including by way of nonlimiting example scrapings from epithelial
surfaces, surgical excision of epithelial tissues, various
biopsies, and surgically resected tissues and organs. In
nonlimiting embodiments, such samples are fixed and embedded in a
supporting material, and tissue slices thereof are prepared in a
microtome or similar instrument. The tissue slices are mounted on
microscope slides. Additionally samples for analysis may originate
from a biopsy, blood, lymph, urine, an effusion fluid, a biological
fluid, a lavage fluid, aspiration fluid, sputum, and a tissue.
[0077] In various embodiments a slide-mounted tissue slice is then
treated with a generic fluorescent dye that stains chromosomes or
nucleic acids with a fluorescent probe having a particular emission
color isolatable by a suitable optical filter. A nonlimiting
example of a generic dye is 4',6-diamidino-2-phenylindole (DAPI).
Staining with DAPI affords a means of identifying the location of
nuclei, or of chromosomes, for the computer driven process of image
capture for further capture of images from FISH probes.
[0078] The tissue specimen is hybridized to a fluorescently labeled
FISH probe whose nucleotide sequence is constructed specifically to
target a gene sequence, or a segment or portion of a gene sequence,
that is specific for an oncogene sought to be targeted. The various
fluorescent labels used in the probes are optically isolatable by
the use of suitable filters and related optical components. The
specificity of the nucleotide sequence ensures that all, or most,
chromosomes in a specimen having the target sequence are in fact
hybridized to the probe, while non-target sequences remain
unhybridized. Hybridization is caused to proceed by heating
sufficiently to denature the target sequence, thereby exposing
single stranded DNA complementary to the probe. The process then
continues by annealing the probe to the exposed single strand, thus
labeling the sequence with the fluorescent label. A worker of skill
in the field of the invention knows specific conditions of solution
ionic strength, buffer composition, temperature, and the like, to
achieve the required hybridization. Following annealing the excess
probe is rinsed away.
[0079] The slide bearing the hybridized specimen is inserted into a
slide-loading cassette that is a component of the automated
microscope system. The system is set into operation, at which point
the slide is caused to be transported from the cassette and placed
on the stage of the microscope. In many embodiments each slide may
bear a code interrogatable by the automated microscope that may
include information such as a specimen identification, and the
identities of any generic chromosome dye, and the various
fluorescent labels on the FISH probes, used with the specimen in
question. Such information guides the automated microscope in
selection of appropriate optical filters and related optical
elements for use throughout the image accumulation process.
[0080] The automated scanning device, such as an automated
microscope, may be configured to scan the biological sample in one
plane, or in more than one plane, such as, for example, two, three
or more planes. By scanning in multiple planes, detection of
abnormal cells, which may be rare in terms of total number of cells
in the sample, may be significantly improved.
[0081] In an embodiment, the probes may make use of FISH probes in
which the fluorescent signal is detected by the detector. It should
be understood that the probes may produce a signal with or without
another input signal, e.g. they may be radioactive, or fluoresce
when stimulated by an activating signal (such as an appropriate
wavelength of light or other electromagnetic radiation). The probes
may be directed to different replication associated
cancer/hyperplasia loci, particular loci associated with a
cancer/hyperplasia. Different fluorescent tags may be associated
with probes to different loci so as to produce different
signals.
[0082] The detector may be selected in accordance with signal(s)
which are to be produced by the tags, e.g. a fluorescence detector
for detecting fluorescent tags, with the detector operatively
configured to permit detection of the particular fluorescent
signals produced by the fluorescent tags.
[0083] Automated analysis may begin by directing the use of a low
magnification of the microscope, using at least the generic dye,
and possibly the probe labels, to identify regions within the
specimen for imaging at a higher magnification. When the computer
software identifies regions of interest at low magnification, it
may direct the automated microscope to interchange objective lenses
and/or filters, and any other optical components, for suitable
image analysis of identified loci at higher magnification based on
emitted light originating from one or another of a fluorescent
label used in a probe. The computer software may then use features
in an image, by way of nonlimiting example, the intensity and
number of FISH-labeled spots, to enumerate such spots arising
within single nuclei. Such an enumeration may provide a resulting
indication of the extent of gene amplification in cells of the
tissue in the specimen being analyzed.
[0084] Reporting may include a simple report of the particular tags
associated with the particular chromosome and/or may comprise an
automatic diagnostic (indicating the type of cancer associated with
the particular hybridization pattern of the chromosomes). In
certain embodiments the automated microscope system automatically
generates a report detailing the findings obtained in the various
images, fields and representations obtained during operation. Such
reports may make use of, or may reference, historical information,
or patient information, already resident in a memory device
associated with the automated microscope.
[0085] A useful system may comprise automatic scanning and
detection in multiple signal channels at once, or in a relatively
short period of time (e.g., less than 1 minute) from one another.
The system may be operatively configured to process each of the
multiple signals in real time, simultaneously or concurrently (or a
mix of the same), to allow for quick detection of chromosomal
regions, and/or regional replications, which are indicative of one
or more particular cancer.
[0086] The vector value as compared to normal specimens may be
selected to be less than a particular threshold, such as less than
about 60, or less than about 40, or less than about 30, or less
than about 20, or less than about 10, or less than about 3, or less
than about 1, or less than about 0.500.
[0087] In a nonlimiting example of an analysis procedure, the
automated method may involve steps such as the following:
[0088] 1. A microscopic specimen is deposited by layering on a
slide a thin section from a paraffin embedded tissue.
[0089] 2. The tissue section is stained using fluorescence in situ
hybridization (FISH) probes for targeted chromosomal loci or
sequences.
[0090] 3. Following FISH probe treatment the slide is scanned using
a desktop scanner at a resolution that may be set at 100, or 200,
or 300, or 400 dots per inch, or more, and the scanned image is
processed in order to identify an area that has been marked by a
pathologist for attention. The digitized information about this
area is passed to an automated fluorescence microscope, such as an
Ikoniscope.TM. microscope system (Ikonisys, Inc., New Haven,
Conn.).
[0091] 4. The slide is loaded in the automated microscope.
[0092] 5. Automated scanning begins by using a low magnification,
such as 2.times., or 4.times., or 5.times., or 10.times.
magnification, or a similar low magnification, for analysis using
the DAPI channel, by which the instrument detects the regions of
the slide that contain nuclei. Typically, scanning is done within
the marked area in step (3).
[0093] 6. Then, using a higher magnification, such as 10.times., or
15.times., or 20.times., or 40.times., or even greater
magnification, the microscope system scans the regions identified
in the previous step. Scanning is performed in the DAPI channel for
the detection of nuclei and then in a channel directed to a
wavelength of light in the range emitted by the fluorescent label
used in the probe, such as an orange channel, for the enumeration
of, for example, orange signals from a FISH probe with a label that
emits orange radiation, and in a green channel for the enumeration
of signals from a FISH probe with a label that emits green
radiation. These provide features of interest, such as nuclei, for
further characterization.
[0094] 7. The positions of features of interest are recorded for
subsequent scanning and verification of signal count in a highest
magnification, such as a 100.times. magnification.
[0095] 8. The automated microscope presents all images collected
during 20.times. and 100.times. scanning to the pathologist for
review and also offers the possibility for subsequent rescanning of
the slides if the pathologist requires review in high magnification
of another slide area.
STATEMENT REGARDING PREFERRED EMBODIMENTS
[0096] While the invention has been described with respect to
preferred embodiments, those skilled in the art will readily
appreciate that various changes and/or modifications can be made to
the invention without departing from the spirit or scope of the
invention as defined by the appended claims. All documents cited
herein are incorporated by reference herein where appropriate for
teachings of additional or alternative details, features and/or
technical background.
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